1. Field of the Invention
This invention relates to a particle measuring device, and more particularly to a device in which, for example, light is applied to separated individual particles to be examined and the optical reaction of the particles to be examined is detected to thereby analyze the nature, structure, etc. of the particles to be examined, or a device for optically measuring the condensation reaction of carrier particles such as sensitized latex and detecting an antigen or an antibody.
2. Related Background Art
Heretofore, in a particle measuring such as a flow cytometer, individual cells have been separated from one another by the method of flowing a high-speed layer flow outside a cell solution devised by Crosland Taylor, whereby it has become possible to obtain information about the individual cells, and much information can be obtained particularly by applying a light beam to the cells to thereby measure the scattered light thereof, and further dyeing particles to be examined in advance with a fluorescent dye and measuring the fluorescence excited and emitted by the application of the light beam. To obtain the scattered light or the fluorescence by minute cells with good accuracy, a light beam of great output which has little noise and is good in condensing property is necessary, and generally a laser light is used. Information such as the size and shape of cells, the diameter and volume of nucleus, and the complexity of the structure in the nucleus is obtained from the scattered light. From fluorescence, it is possible to find the amount of DNA and the amount of RNA by the dyeing of DNA and RNA. Particularly, by coupling the fluorescent coloring matter to an antigen or an antibody, causing it to react to the antigen or the antibody and measuring the fluorescence by a flow cytometer, quantitative measurement of a particular antigen or antibody on the surface of a cell becomes possible. In these cases, the light beam applied must be of a wavelength which excites the fluorescent coloring matter.
However, in the measurement of scattered light by the flow cytometer, the wavelength of the irradiating light has been uniform and new particle analysis data could not be obtained. Also, in fluorescence measurement, the irradiating light beam must be one which excites the fluorescent coloring matter, but the fluorescence coloring matter used is limited to fluorescein, rhodamine or umbelliferon. For these limited fluorescent coloring matters, a short wavelength gas laser such as Ar.sup.+ laser (488 or 515 nm), He-Cd.sup.+ laser (442 or 325 nm) or N.sub.2 laser (337 nm) or a coloring matter laser excited by a short wavelength laser is used as an irradiating light beam. Such a short wavelength laser or a coloring matter laser becomes bulky when a great output is to be obtained, and also lacks in stability, and this has led to the disadvantage that a particle measuring device using it also is bulky and complex and lacks in stability.
Also, where a plurality of fluorescent coloring matters of different absorbing wavelengths are used at one time, if an exciting light of a single wavelength is used, the intensity of fluorescence will be remarkably reduced and in an extreme case, no fluorescence will be emitted. Therefore, it has been necessary to prepare a plurality of laser sources of wavelengths suitable for exciting the respective fluorescent coloring matters, but in such case, the device becomes more bulky and more expensive, and the optical system for irradiating particles to be examined also becomes complex. Devices of such a construction are described, for example, in U.S. Pat. No. 3,826,364 and U.S. Pat. No. 4,284,412.
As another particle measuring device, there is known a device for measuring immunoreaction by the use of carrier particles such as latex. A suspension with a sample to be examined (for example, serum) including antigens or antibodies added to a suspension in which insoluble carrier particles (for example, latex particles) sensitized by a particular antibody or antigen are suspended at a predetermined concentration is prepared and an irradiating light is applied thereto. If at that time, the latex particles in the suspension are in their dispersed state, the light of much longer wavelength than the particle diameter is transmitted without being much affected by the presence of the latex particles, as shown in FIG. 15A of the accompanying drawings. That is, transmitted light of great intensity is obtained. However, when said sensitized latex particles are coupled together by the reaction of the antigen or antibody to form particle lumps of a large particle diameter and the particle diameter of the condensed particle lumps becomes approximate to the wavelength of the light, the light is scattered by the particle lumps as shown in FIG. 15B of the accompanying drawings and the intensity of transmitted light decreases. A reaction speed analyzing method of measuring and analyzing the concentration of the suspension from the reaction speed catching the variation in this intensity of transmitted light with time, and a reaction terminal analyzing method of measuring and analyzing the intensity of transmitted light or the intensity of scattered light in the suspension after the reaction terminates are generally known. Thereby, measurement of the amount of particular antigen or the amount of particular antibody in the sample to be examined has become possible and immunological diagnosis has been done.
As an example, a latex particle suspension of a predetermined concentration which is sample liquid is stored in an optical cell 54 which is a transparent container, as shown in FIG. 13 of the accompanying drawings, and a laser light is applied from a laser source 51 toward. The intensity of transmitted light therethrough is detected by a photodetector 56 and the absorbance thereof is found, and the size and amount of the reacting mixture in the sample liquid are detected, whereby the condensed state of the latex particles can be judged, and the amount of the antigen or antibody which is the object can be quantified.
However, where the size of the condensed lumps of latex particles is to be detected by the absorbance of the latex particle suspension as in the above-described example of the prior art, the absorbance increases linearly as shown in FIG. 14 of the accompanying drawings as the particle diameter of the latex particle lumps becomes larger, but where the wavelength of the irradiating light is a short wavelength such as 400 nm or 600 nm, when the latex particles exceed a predetermined size, the graph of the absorbance is reduced, and two particle diameters correspond to the absorbance found from the intensity of transmitted light, and it has been impossible to judge which particle diameter is right. So, where use is made of a light of such a long wavelength that the graph of the absorbance is not reduced, for example, a light of wavelength 800 nm, if a certain particle diameter (about 1.0 .mu.m) is exceeded, the variation in the absorbance will become small relative to the variation in the particle diameter and measurement sensitivity will become bad.
Therefore, to improve accuracy, it is preferable to prepare a plurality of laser sources of different wavelengths, but as noted previously, this has led to the problems of the bulkiness and expensiveness of the device.